Process of producing dextran



ma Jun PROCESS OF PRODUCING DEXTRAN Earl R. Kooi, La Grange, Ill.,assignor to Corn Products Refining Company, New York, N. Y., acorporation of New Jersey V M.

No Drawing. Application April 7, 1954, Serial No. 421,694

20 Claims. (Cl. 195- -32) is grown on a substrate containing as oneofthe constituents a carbohydrate, polyhydric alcohol, or a similar sourceof organic carbon-containing material, three enzyme systems areproduced, (1) dextran-dextrinase, which converts dextrins to dextran,(2) an amylase, which hydrolyzes glucose polymers to glucose, and (3) aglucose oxidase or glucose dehydrogenase which converts glucose togluconic acid. Thus, when it is desired to prepare dextran by a processinvolving (1) fermentation of a carbohydrate material, for example, forthe accumulation of dextran-dextrinase followed by (2') an enzymeconversion of a partially hydrolyzed amylaceous material for theaccumulation of dextran, both of the other enzyme systems are operativeduring this second period. The result is that considerable quantities ofgluconic acid accumulate during the enzyme conversion period, and unlessthis acid is continuously neutralized, the pH value of the enzymeconversion liquor falls below the optimum value for the accumulation ofdextran.

The main object of this invention is to prevent the formation ofgluconic acid in such process. This is accomplished by the addition tothe enzyme digest of a material which specifically inhibits the glucoseoxidase or glucose dehydrogenase, but causes no inhibition of thedextrandextrinase.

In carrying out the invention an organism such as Acetobacter capsulatumor Acetobacter yiscosum is grown for an initial period on a substrateconducive to the formation of dextran-dextrinase. To the culture liquor,adjusted to the pH optimum for deXtran-dextrinase activity, there isthen added a partially hydrolyzed amylaceous material, such as a starchhydrolyzate, having a D. E. value of about 5 to about 65 and a material,such as toluene, which specifically inhibits the formation of gluconicacid by enzymes from the above organism. The mixture is then incubatedunder conditions conducive to dextran formation. The substrateaforementioned may be a polyhydric alcohol or a partially hydrolyzedamylaceous material or the like, and although the substrate on which theorganism is grown is not to be considered a part of this invention, itshould be appreciated that the three enzyme systems named above areproduced by these two organisms whether the carbon source of thesubstrate is a partially hydrolyzed amylaceous material or a polyhydricalcohol or other suitable material.

ited States Patent 0 The process of my invention will now be describedin further detail with particular reference to partially hydrolyzedstarch as both the substrate for the formation of dextran-dextrinaseand, also, as the raw material for the formation of dextran. Starch ishydrolyzed as with acid "in conventional mann'eruntil the D. E.va1ue'(dextrose 2,801,205 Patented July 30, 1957 equivalent, i.e., percent ofreducing sugars, on dry basis, calculated as dextrose) is within therange of about 5 to about 65. The pH of the resultant starch hydrolyzateis adjusted to 4.5 to 5.5, and then the hydrolyzate is skimmed orfiltered. Thereafter, the solids content is adjusted to about 5 percentand there is added to the concentrated liquor about 0.5 gram per ml. ofan organic nitrogenous material, such as dehydrated. yeast extract, cornsteep liquor, or the like. The pH of the liquor is then adjusted to 5.5to 6.8 and the liquor is sterilized in known manner, e. g., at about C.for 20 minutes. Next the resultant fermentation medium is cooled toabout 25 to 30 C. and thereafter it is inoculated with a suitableculture grown 10 to 48 hours on the same medium as heretofore describedin amount within the range of about 2 to about 20 percent by volume offermentation medium. The medium is agitated and aerated untildextran-dextrinase activity reaches a substantially maximum value, forexample about 10 to 16 hours. There is then added to the culture liquora material which inhibits .the enzymatic formation of gluconic acid butdoes not inhibit the dextran-dextrinase. There may also be added anadditional quantity of starch hydrolyzate. The liquor is then incubatedunder conditions conducive to the formation of dextran by thedextran-dextrinase, as for example at pH 4.5 and 30 to 35 C.

I have previously discovered that the rate of dextran formation fromstarch hydrolyzates can be markedly increased by agitation and aerationof the culture liquor. Further that the rate of dextran production andthe concentration at which the substrate can be efficiently con vertedto dextran can be' eifectively increased by starting the fermentation ofa carbohydrate concentration of not more than 10 grams per 100 ml. andadding a concentrated solution of the carbohydrate material after activefermentation has begun. An additional means of insuring rapid dextranformation at high carbohydrate concentrations is to maintain the pH ofthe culture liquor at about 5.5 to 6.8 for the first 10 to 24 hours andthereafter at about 3.5 to 5.0 until the fermentation is completed.These discoveries are described in my co-pending application, Serial No.328,154, filed December 26, 1952.

If an inhibitor for the enzymes producing gluconic acid is not added,the pH of the liquor drops to a value which is not optimum for theaction of the dextrandextrinase. For example, comparative rates ofdextran formation by the dextran-dextrinase enzyme at diiferent pHvalues are as follows:

Rate of dextran Although the pH during the enzymatic formation ofdextran may be maintained at the optimum value by continuousneutralization of the gluconic acid, in either case in the absence ofthe inhibitor considerable quantities of gluconic acid accumulate. Bymeans of the present invention, it is possible to maintain stable,optimum pH during the course of the enzymatic formation of dextranwithout recourse to' additional neutralization. Further,

the invention is also advantageous in that the prevention of theformation of gluconic acid results in easy recovery of both dextran andthe residual, non-dextran carbohydratesince only two materials need beseparated. There is no loss of carbohydrate due to formation of gluconicacid.

The invention is particularly applicable to enzymatic conversion ofpartially hydrolyzed materials, e. g. any starch, to dextran, whereinthe enzymes are obtained from cultures of Acetobacter and wherein theenzyme mixture contains, in addition to the dextran-dextrinase enzyme,an enzyme system which converts glucose to gluconic acid.

The following examples will further illustrate the invention but itshould be understood that they are typical and informative and not inany way limiting the invention.

Example 1 Acetobacter capsulatum was grown for 17 hours on a mediumcomposed of a 17 D. E. acid hydrolyzate of corn starch together withyeast extract, as above described. The culture liquor was then adjustedto a pH 4.5 and divided into two portions. To each was added sufiicientof a 17 D. E. acid hydrolyzate of corn starch having a pH of 4.5 tobring the total concentration to 13 grams of dry substance per 100 ml.To one portion was added one percent toluene, by volume. Both portionswere agitated constantly at 30 C. Following are the results obtained:

It is readily seen from the above example that toluene completelyinhibits the formation of gluconic acid and thus results in themaintenance of a constant pH value throughout the enzymatic formation ofdextran. Further, the addition of toluene even in amounts as great aspercent by volume has no detrimental effect on the formation of dextran,although there is no advantage in using more than about one percent, byvolume.

Example 2 Other materials which behave similarly to toluene when usedunder the conditions described in Example 1, i. e., inhibit theformation of gluconic acid without inhibiting dextran-dextrinaseactivity are:

Lowest Effective Concentration, Grams per 100 m1.

Material Benzene 0.50.

about 1.0.

yr about 0.1. Monobutyl-rneta-cresol about 0.025.

Amounts of inhibitors greater than listed above may be used, but thereis no particular advantage in the use of more than the values givenabove insofar as prevention of gluconic acid formation is concerned.

Example 3 The following example shows that toluene (one percent byvolume) is effective in the prevention of gluconic acid formation in theenzymatic formation of dextran from various starch hydrolyzates byenzymes derived from Acetobacter capsulatum. The initial pH of each 4 ofthe substrates was 4.4 to 4.5. The time of the enzyme conversion in eachcase was hours.

Percent D. E. of Hydrol- Method of Hydrolysis of Starch Yield of yzateDextran 28 24 20 t 35 malt enzyme 15 acid to 17 D. E. plus malt. 15acid... 20 do 13 do 10 acid to 17 D. E. plus fungal enzyme 3 ar-id 6acid to 17 D. E. plus fungal enzyme 3 No gluconic acid was produced inany of the above samples in 100 hours of incubation as determined bydecrease in pH, the pH remaining the same.

I claim:

1. A process for producing dextran which comprises converting thedextrin, in a partially hydrolyzed amylaceous material, to dextran, bymeans of dextran-dextrinase derived from Acetobacter, in contact with aninhibitor for glucose dehydrogenase from the group consisting ofbenzene, alkyl benzene and hydroxy alkyl benzene.

2. Process of claim 1 wherein said hydrolyzed amylaceous materialcomprises starch hydrolyzed to a D. E. value within the range of about 5to about 65.

3. Process according to claim 2 wherein said inhibitor is toluene inamount of at least about 0.25 gram per 100 ml. of substrate.

4. Process according to claim 2 wherein said inhibitor is benzene inamount of at least about 0.50 gram per 100 m1. of substrate.

5. Process according to claim 2 wherein said inhibitor is xylene inamount of at least about 1.0 gram per 100 ml. of substrate. 1

6. Process according to claim 2 wherein said inhibitor is thymol inamount'of at least about 0.1 gram per 100 ml. of substrate.

7. Process according to claim 2 wherein said inhibitor ismonobutyl-meta-cresol in amount of at least about 0.025 gram per 100 ml.of substrate.

8. Process according to claim 2 wherein said dextrandextrinase isderived from Acetobacter capsulatum.

9. Process according to claim 2 wherein said dextrandextrinase isderived from Acetobacter viscosum.

10. A process for producing dextran which comprises fermenting a mediumcomprising a source of nitrogen and a partially hydrolyzed amylaceousmaterial with an organism from Acetobacteriaceae untildextran-dextrinase activity reaches substantially the maximum,thereafter adding an organic compound which is an inhibitor for glucosedehydrogenase from the group consisting of benzene, alkyl benzene andhydroxy alkyl benzene, and allowing said dextran-dextrinase to convertthe substrate to dextran.

11. Process according to claim 10 wherein said hydrolyzed amylaceousmaterial comprises starch hydrolyzed to a D. E. value within the rangeof about 5 to about 65.

12. Process according to claim 10 wherein the pH is maintained withinthe range of 5.5 to 6.8 during the fermentation and within the range of3.5 to 5.0 during the enzyme conversion.

13. Process according to claim 11 wherein said inhibitor is toluene inamount of at least about 0.25 gram per 100 ml. of substrate.

14. Process according to claim 11 wherein said inhibitor is benzene inamount of at least about 0.50 gram per 100 ml. of substrate.

7 15. Process according to claim 11 wherein said inhibitor is xylene inamount of at least about 1.0 gram per 100 m1. of substrate.

16. Process according to claim 11 wherein said inhibitor is thymol inamount of at least about 0.1 gram per 100 ml. of substrate.

17. Process according to claim 11 wherein said inhibitor is monobutylmeta-cresol in amount of at least about 0.025 gram per 100 ml. ofsubstrate.

18. Process according to claim 11 wherein the dextrandextrinase isderived from Acetobacter capsulatum.

19. Process according to claim 11 wherein the dextrandextrinase isderived from Acetobacter viscosum.

20. Process according to claim 11 wherein the medium is agitated andaerated during the formation of the deXtran-dextrinase References Citedin the file of this patent UNITED STATES PATENTS 2,689,816 Kooi Sept.21, 1954 5 OTHER REFERENCES

1. A PROCES FOR PRODUCING DEXTRIN WHICH COMPRISES COVERTING THE DEXTRIN,IN A PARTIALLY HYDROLYZED AMYLACEOUS MATERIAL, TO DEXRAN, BY MEANS OFDEXTRAN-DEXTRINASE DERIVED FROM ACCTOBACTER, IN CONACT WITH AN INHIBITORFOR GLUCOSE DEHYDROGENASE FROM THE GROUP CONSISTING OF BENZENE ALKYLBENZENE AND HYDROXY ALKY BENZENE.